17-ERACoBioTech: Fabrication of hierarchically organized multi-functional heterogeneous biocatalysts

由酶催化的化学合成有助于建立以更清洁、更快和更安全的化学反应为支撑的现代化学。特别是，溶液中的无细胞合成生物学(或系统生物催化)目前正在成为使用全细胞合成生物学的一种有吸引力的替代方案，因为分离的酶不存在基因组水平的调控限制，并且化学通量的增强对系统生存的影响可以忽略不计。然而，它在工艺效率和成本效益方面都存在重大问题，因为这些可溶体系往往达到较低的化学产率，非常不稳定，而且其可重复使用性相当有限。为了克服这些限制，该提议旨在通过蛋白质支架在固体和多孔材料的纳米尺度上组装多酶系统，以保证功能模块的分层和空间组织。这种固定化的多酶串联体系将被用作多相多功能生物催化剂，在一锅中将可再生的可再生原料转化为omega-氨基酸，并进行原位辅因子再生。这种多相生物催化剂的制造和开发将通过以下步骤来实现：i)以生物油和二醇为原料设计合成omega-氨基酸的人工途径，ii)设计模块化蛋白质(共识四肽重复序列：CTPR)作为在纳米尺度上组织多酶系统的支架单元，iii)将多酶系统体外组装到CTPR支架上，iv)将这种多酶组件固定在固体颗粒上，V)使用分级组织的多功能多相生物催化剂高效和可持续地生产长和短的欧米伽-氨基酸6)10 L在工业相关条件下放大该过程，以及vii)制造一个基于表达不同多酶系统组装的DNA质粒的模块试剂盒和一个允许基于支架多酶系统的不同功能模块的固相组装的模块试剂盒。这些新平台将开启一种创新工具，为使用可再生原材料的高附加值分子的化学制造建立可持续的途径。通过结合蛋白质工程、表面化学和蛋白质固定化工具，将不同的酶作为功能模块与工程蛋白支架和作为异质底盘的多孔材料进行合理集成。研究团队在这些领域提供了坚实的多学科背景，这使该团队完全有资格在化学和生物之间的前沿开展这一项目。此外，研究联合体由不同的研究机构主办，这些机构提供合适的研究环境(三个学术合作伙伴：CIC BioaGUNE、诺丁汉大学和波鸿鲁尔大学，以及一个工业合作伙伴：生物分析)，以成功应对主要挑战并实现该项目的主要目标。

Chemical synthesis catalyzed by enzymes is contributing to establish a modern chemistry supported on cleaner, faster and safer chemical reactions. In particular, cell-free synthetic biology (or systems biocatalysis) in solution is currently emerging as an attractive alternative to synthetic biology using whole cells because isolated enzymes do not present regulation constraints at genomic level and the intensification of the chemical fluxes do negligible effect on the system subsistence. However, it presents major issues in terms of both process- and cost-efficiency because these soluble systems often reach low chemical yields, are notably unstable and their re-usability is rather limited. In order to overcome these limitations, this proposal aims to assemble multi-enzyme systems at the nanoscale of solid and porous materials aided by protein scaffolds that guarantee the hierarchical and spatial organization of the functional modules. This immobilized multi-enzyme cascade will be utilized as heterogeneous multi-functional biocatalyst to transform renewable raw materials into omega-aminoacids, in one-pot with in situ cofactor regeneration. The fabrication and exploitation of such heterogeneous biocatalyst will be achieved by i) engineering the proposed artificial pathway for the synthesis of omega-aminoacids using bio-oils and diols as raw materials, ii) engineering a modular protein (consensus tetratricopeptide repeat: CTPR) as scaffolding unit to organize the multi-enzyme system at the nanoscale, iii) in vitro assembling of multi-enzyme systems onto the CTPR scaffolds, iv) immobilizing such multi-enzyme assemblies on solid particles, v) using the hierarchically organized multi-function heterogeneous biocatalysts for the efficient and sustainable production of long and short omega-aminoacids vi) 10 L scale-up the process under industrially relevant conditions and vii) manufacturing of one modular kit based on DNA plasmids that express assemblies of different multi-enzyme systems and one modular kit that allows the solid-phase assembly of different functional modules based on scaffolded multi-enzyme systems. These new platforms will open an innovative tool to build sustainable pathways for chemical manufacturing of high added value molecules using renewable raw materials. The rational integration of different enzymes as functional modules with an engineered protein scaffold and a porous material as heterogeneous chassis will be addressed by combining protein engineering, surface chemistry and protein immobilization tools. The research team presents a solid and multidisciplinary background in those areas, which fully qualifies this team to carry out this project at the frontier between the chemistry and the biology. Moreover, the research consortium is hosted in different research institutions that provide a suitable research environment (three academic partners; CIC biomaGUNE, University of Nottingham and Ruhr Universität-Bochum, and one industrial partner: Bioassays) to successfully address the major challenges and meet the main objectives of this project.